Capillary Gel Electrophoresis (CGE) is a sensitive method for analysis and identification of biological molecular systems. CGE is a relatively new analytical separation technique that can be applied to the analysis of a wide variety of compounds that provide for improved resolution over other existing techniques. Its use for increasing the rate at which DNA sequencing can be performed has been of particular interest. Because of its sensitivity, the technique is gaining acceptance in many laboratories and manufacturing operations of drug and chemical manufacturers worldwide. However, the instrumentation that is being used to produce the data using this technique is still relatively inefficient, complex and expensive. Although these systems can appear physically different from each other, they all contain the basic functional blocks required for this type of analysis. Each has a method of holding the capillaries, injecting samples therein, transmitting and collecting light, detecting a fluorescent signal from each sample being measured that is induced by the incident light energy, applying voltage to the capillaries, and outputting the collected data in some form.
What these systems generally suffer from is that the techniques involve equipment that is not cost effective for high volume manufacturing, and consequently does not permit widespread use of this important analytical technique. The performance of a single capillary system depends on the method of sample excitation and on the signal collecting optics. In multi-capillary systems precise alignment of delivery, collection and sample assemblies can be difficult. In free beam systems this has been done by visual inspection of reflected or transmitted laser light.
There is a continuing need for improvements in systems for performing optical measurements of biological samples that are readily manufacturable, have low maintenance costs and provide for fast accurate analysis of a large number of samples.
This invention relates to a system and method for delivering light to chemical or biochemical samples using an aligned optical fiber delivery system that couples light from a light source with an array of sample channels. Light from the samples is collected and detected for data analysis, presentation, and storage. The optical signal collection is accomplished by a second optical fiber system. In a preferred embodiment, the delivery and collection optical fiber systems are mounted and permanently aligned on a mounting structure such that each channel or capillary is in the same plane as the delivery fiber and collection fiber for that capillary. The delivery and collecting fibers can be selected with respect to their core sizes and numerical apertures to satisfy the particular application requirements. The collecting fiber largely filters out the excitation light, reducing the detection noise and improving the detection sensitivity. A multi-mode fiber can be used for this purpose. In an optical fiber CGE delivery and collection system, the collecting fiber fulfills the role of a spatial filter, lens and a light guide. The two fibers and the capillary are co-planar, enabling a practical and inexpensive method of fabricating a multichannel assembly. The spatial filtering of the undesired, noise-generating, excitation light in the collecting fiber has improved performance over free beam systems where reflections dominate the fluorescence signal.
This fiber optical system presents a number of advantages over the free beam technology used in existing systems. There are no optical components other than fibers, thereby reducing cost, complexity and size. Also, the geometry reduces the amount of excitation light reflected back to the collecting fiber, improving signal to noise ratio. Another advantage of this fiber system is simplification of multicolor detection in comparison with free beam optics where the focal length of lenses, or deflection angles are wavelength sensitive, making simultaneous focusing of different colors difficult. This is not the case in a fiber based system where the emitted light fills substantially the same cone of light at the fiber output and input.
A preferred embodiment of the invention pertains to all fiber systems where the fiber and capillary assemblies are fabricated by affixing them on precision planar surfaces. This relies on highly precise features or grooves formed on a silicon wafer or substrate, for example, by well known micromachining techniques. A large number of capillaries can be precisely aligned and measured with this system, thereby substantially increasing the rate of sample analysis. Arrays of capillaries or channels can be manufactured in multiples of 4 or 8, including 16 or 32, for example. Features are accurate to within 10 microns or less to provide the accurate positioning necessary to achieve the desired measurement accuracy.
Another preferred embodiment of the invention relates to the use of the grooves or channels in the substrate instead of the capillary tubes to confine the gel. A quartz window can be attached to the grooved substrate to provide an optical window for all of the channels in an array. The window can also have a groove to provide a symmetric cross-section to the channel.
Alignment features can also be incorporated in the substrates. An optical alignment system is described here where an alignment accuracy of less than 10 microns, and preferably of about 1 um is employed. This method makes use of the precise geometry of the fiber and capillary assembly substrates.
The registration feature can be a single or a multiple groove structure depending on the method used. The optical registration technique involves detecting a change in surface reflectivity when a fiber tip moves over a groove or a similarly reflecting feature in the reflecting surface. If the fiber position is fabricated precisely with the reflecting feature the change of reflectivity indicates the point of registration.
Another preferred embodiment of the invention includes a system and method for positioning an optical fiber relative to a measurement cell such as a capillary tube. In this system light emitted by an optical fiber is reflected by the capillary surface, for example, and the intensity of the sensed reflected signal is compared to a reference value. The comparison is used to stop the motion of the optical fiber system when it is correctly positioned. A feedback control system can be used to automatically position either the optical fiber system, or the capillary system, relative to the other.
Another preferred embodiment of the invention relates to the use of a light source which emits a plurality of wavelengths. For example, a plurality of lasers can provide multiple color excitation. Multiple lasers are optically coupled to each capillary or channel. Thus, multiple lasers feed a single fiber that carries the light to the sample-carrying capillary. Each source can be modulated at a different modulation frequency so that the combined beams contain multiple modulation frequencies. Further, each source can be modulated separately which minimizes cross talk during coherent detection. A single detector can detect multiple emission peaks simultaneously. The detected fluorescence signal generates photocurrent that contains multiple frequencies. The ratio of the amplitudes depends on the fluorescent label excited by the light beam. This embodiment provides a multi-analyte detection capability and expands the use of the system of the present invention to perform a variety of analysis and DNA sequencing operations.
Another preferred embodiment of the invention includes using a light emitting device such as a light emitting diode (LED) as the light source. The LED light source can provide the user with the option of using a variable intensity for each LED. Thus, the intensity of each LED signal can be modulated at a different frequency and detected using an electronic filter for each detected signal that is tuned to the frequency of the corresponding LED. LEDs with different emission wavelengths can be used for different channels to match the absorption bands of different dyes that can be used in the different channels.
In a preferred embodiment, a plurality of semiconductor lasers can be used to irradiate the channels. For certain applications lasers can provide more efficient pumping of the dye and thereby improve sensitivity. For example, III-V semiconductor materials can be used to fabricate a solid state array of lasers emitting in the visible or near infrared range, and preferably between 400-500 nm that are matched to the excitation band of a selected dye. Gallium nitride based lasers emitting at a center line of 417 nm are available for this application, for example. Such a laser array can be optically coupled using a fiber array or with a lens array as described herein. InGaN based lasers can be used at longer wavelengths. Other regions of the visible or near-infrared up to 850 nm can also be used with LEDs or semiconductor lasers based on GaAs or InGaAs.
A preferred method using two or more lasers emitting at different excitation wavelengths to illuminate each capillary. For example, three lasers emitting at three different wavelengths can be used to measure four different labels having different emission peaks. Each capillary in this four color analysis system can have three or four lasers that are optically coupled therewith using the optical systems described herein.